a. Field of the Invention
The invention relates to spectrofluorometers, and more particularly to an improved spectrofluorometric method and apparatus which minimizes the effect of background or competing fluorescence.
B. Prior Art
In fluorescent spectroscopy, a sample containing a material of unknown quantity, i.e. target molecules, is placed in a holder. The sample may be in the form of a liquid solution or a solid on a substrate such as filter paper. The sample is then exposed to radiation of known spectral distribution, characteristically a restricted bandwidth of light. The distribution of excitation radiation is within the excitation spectrum of target molecules and preferably near the maximum. The emission spectrum following irradiation is at longer wavelengths and is characteristic of the target molecules. Its intensity is measured to indicate the quantity of target molecules.
One problem with present spectrofluorometers is that their sensitivity is limited due to noise in the excitation and detection systems and secondly, due to competing fluorescence from ambient substances, such as from substrate materials, sample containers, particles in the air, other fluorescing species in the sample, reagents and the like.
Prior to the filing of this patent application, a pulsed dye laser has been reported for use in a spectrofluorometer. See the article entitled "New Type of Spectrofluorometer with a Tunable Laser Source and Unique Optical System" by D. C. Harrington and H. V. Malmstadt, Analytical Chemistry, Vol. 47, No. 2, February 1975, p. 271-276.
Use of a pulsed laser to stimulate fluorescent emission from target molecules improves the signal to noise ratio to a certain extent. However, while the signal to noise ratio is enhanced, the problem of competing fluorescence is still present. When the amount of target material is very small, perhaps a few parts per billion or less, the fluorescence of the target molecules may not be detected because it may be weaker than the combined fluorescence from all background sources.
It is therefore an object of the invention to provide a method for detecting very small quantities of target molecules in the presence of competing background fluorescence.
Another object is to devise an improved spectrofluorometer for detecting very small quantities of target molecules in the presence of competing background fluorescence.